The non-pathogenic human dependoviruses, adeno-associated viruses (AAV), infect either proliferating or post-mitotic cells. Since all the AAV and helper virus genes can be provided in trans, the entire AAV genome may be replaced with the gene of interest. The resulting recombinant AAV (rAAV) may then be produced transiently in cell culture and the rAAV particles concentrated and purified based on physical properties of the virion. The prototype for AAV is serotype 2 (AAV2). Until recently, all characterizations of AAV genetics, molecular biology, and vector development have been performed with AAV2. We have cloned, sequenced and partially characterized AAV types 4 and 5 in order to define activity domains of the non-structural Rep proteins and develop vectors with different tissue specificities than AAV2. AAV type 4 - AAV 2 and AAV4 rep ORF sequences are 90% identical at the amino acid level. The divergent sequences appear in regions that are tolerant of mutation. However, the sequence of the AAV4 capsid protein genes were only 62% identical to AAV2. The difference in capsid proteins resulted in overlapping but distinct tropism. In order to determine whether the different tropism resulted from utilization of different receptors, cells were co-infected with a reporter virus and a non-scoring "cold AAV2". A 72-fold excess of cold AAV2 was needed to inhibit AAV4-lacZ transduction 50%. Whereas rAAV2-lacZ was competed with approximately a 10-fold excess of cold AAV2. Furthermore, trypsin treatment of cells reduced the percent transduction of rAAV2-lacZ by 80%, yet rAAV4-lacZ transduction was virtually unaffected. The competition experiments and sensitivity to trypsin are strong indicators that the uptake mechanisms of AAV2 and AAV4 are different. AAV type 5 - Another serotype that had not been characterized is AAV type 5. We cloned the virus genome and determined the sequence. The Rep open reading frames of types 2 and 5 are 67% identical and the capsid open reading frames are 60% identical. Thus, serotypes 2 and 5 are the most divergent of the known AAVs. Sequence alignments revealed that AAV 5 is approximately equally related to goose or moscovy duck parvovirus as to AAV 2. Functions of the Rep polypeptides appear to be conserved, but specificity for endonuclease activity is altered relative to type 2. The uptake mechanism of AAV 5 seems to be distinct from AAV 2 based on competition with type 2 virus. AAV 2 has been shown to bind heparan sulfate proteoglycans and soluble heparin neutralizes adsorption to the cell. Neither AAV 4 nor AAV5 are neutralized with soluble heparin.